1-fluoro, 4-fluoro, and 1,4-difluoro anthracycline anticancer antibiotics

ABSTRACT

Compounds represented by the formula (I) ##STR1## wherein R is hydrogen or hydroxyl, one of X and Y is fluorine and the other is hydrogen or both X and Y are fluorine, and Su is a hydrogen atom or a sugar moiety; where Su is a sugar moiety, the compounds are useful anticancer antibiotics; pharmaceutical preparations containing the antibiotics and a method for inhibiting the growth of mammalian tumors are also disclosed.

BACKGROUND OF THE INVENTION

Anthracycline antibiotics including doxorubicin, daunorubicin, andcarminomycin are important chemotherapeutic agents in the treatment of abroad spectrum of neoplastic conditions including acute myeloblastic andlymphoblastic leukemias. Doxoruoicin (also known as Adriamycin) is thesubject of U.S. Pat. No. 3,590,028 and is a prescribed antineoplasticagent used in a number of chemotherapeutic treatments. Specifically,doxorubicin and daunomycin have the formula: ##STR2## with the compoundbeing doxorubicin when R is --OH and daunomycin when R is --H.

In view of the proven effectiveness of known anthracyclines in thetreatment of cancer, efforts have been undertaken to develop less toxicderivatives which can be administered in high, more effective dosageswith greater frequency.

U.S. Pat. No. 4,046,878 to Patelli et al. discloses daunomycin analoguessubstituted at the 1-position and/or the 4-position with chlorine orbromine.

SUMMARY OF THE INVENTION

The present invention relates to compounds represented by the formula(I) ##STR3## wherein R is hydrogen or hydroxyl, one of X and Y isfluorine and the other is hydrogen, or both X and Y are fluorine, and Suis a sugar moiety or a hydrogen atom.

More particularly, the present invention relates to anthracyclineantibiotics of the formula (I) wherein S is a sugar moiety and S isrepresented by the formula (II) ##STR4## wherein one of R¹ and R² ishydrogen and the other is hydroxy, acyloxy, or a halogen atom selectedfrom fluorine, chlorine, bromine or iodine; one of R³ and R⁴ is hydrogenand the other is amino; and R⁵ and R⁶ are hydrogen. Suitable acyloxygroups have 2 to 4 caroon atoms, the most typical example beingO-acetyl.

Still more particularly, tne present invention is directed to compoundsof the formula (I) wherein S is dacosamyl; namely, R₁ is hydroxy, R₂ ishydrogen, R₃ is hydrogen, R⁴ is amino and R⁵ and R⁶ are hydrogen.

The present invention also provides pharmaceutical preparationscontaining the aforesaid compounds in therapeutically effective amountsin suitable carriers.

The compounds of the present invention have been shown to be effectiveagainst lymphoblastic leukemia in the P388 leukemia screen. In addition,it is also anticipated that the compounds of the present invention willbe effective in the treatment of other tumors such as ascitic tumors,solid tumors sucn as sarcoma 180, and L1210 leukemia.

Certain of the compounds of the present invention are also available inthe form of acid addition salts. Suitable salts include hydrochlorides,citrates, formates, etc.

Therapeutic compositions containing the novel compounds of the presentinvention as active agents can oe prepared by dispersing or dissolvingthe compound in any pharmaceutically acceptable, non-toxic carriersuitable for the desired mode of administration. Therapeuticcompositions of the present invention may be administered parenterallyby intravenous, intraperitoneal, or other conventional injection ororally in some cases. Preferably, the carrier is an aqueous mediumbuffered to pH 7.2 to 7.5, the physiological range. Any suitableconventional buffer can be used such as tris phosphates, bicarbonates orcitrates. If desired, saline solution can be used, with pH adjustmentand buffering. Optimal dosages may vary over a broad range fromapproximately 0.1 to 20 mg/kg of body weight depending upon theparticular compound and mode of administration employed.

The present invention also provides a metnod for inhibiting the growthof mammalian tumors comprising administering a therapeutically effectiveamount of the aforesaid compounds to animals afflicted with such tumors.

DETAILED DESCRIPTION OF THE INVENTION

Examples of antibiotics in accordance with the present invention are4-demethoxy-4-fluorodaunomycin, 4-demethoxy-4-fluorodoxorubicin,4-demethoxy-1-fluorodaunomycin, 4-demethoxy-1-fluorodoxorubicin,4-demethoxy-1,4-difluorodaunomycin, 1,4-difluorodoxorubicin, andderivatives of these antibiotics wherein the sugar moiety is substitutedwith a halogen atom selected from the group consisting of fluorine,chlorine, bromine or iodine. The latter compounds include3'-deamino-4-demethoxy-3',4'-di-O-acetyl-4-fluoro-2'-iododaunomycin and3'-deamino-4-demethoxy-3',4'-di-O-acetyl-4-fluoro-2'-iododoxorubicin;3'-deamino-4-demethoxy-4-fluoro-2'-iododaunomycin and3'-deamino-4-demethoxy-4-fluoro-2'-iododoxorubicin;3'-deamino-4-demethoxy-1-fluoro-2'-iododaunomycin and3'-deamino-4-demethoxy-1-fluoro-2'-iododoxorubicin and4-demethoxy-4'-deoxy-4-fluoro-4'-iododaunomycin and4-demethoxy-4'-deoxy-4-fluoro-4'-iododoxorubicin. A particularlypreferred class of compounds are the 4'-epi derivatives of theaforementioned compounds.

The compounds of the present invention can be prepared from aglyconesobtained by the synthesis outlined in U.S. patent application Ser. No.692,584, filed Jan. 18, 1985 (which is hereby incorporated herein byreference). They can be prepared by reacting a cyanophthalide anion ofthe formula (III) ##STR5## wherein X and Y are defined as above with aquinone monoketal of the formula (IV) where at least one of W₁ and W₂ isa protecting group and the other is hydrogen. The resultinganthracyclinone can be coupled with an appropriate sugar using theapproach of Acton et al., J. Med. Chem., 17, 659 (1973).

The required cyanophthalides IIIa-c were obtained by two general routes.The first involves ortho-metallation of the dimethylacetal (V) followedoy reaction with carbon dioxide and hydrolysis to afford thehydroxyphthalide (VIa). ##STR6##

The above chemistry was less convenient for the preparation ofhydroxyphthalides (VIb,c) and an alternative route was employed forthese latter two compounds. The required oxazolines (VIIb,c), preparedby standard methods [see J. W. Conforth, Heterocyclic Compounds 5, 386(1957); A. I. Meyers and D. L. Temple, Journal of the American ChemicalSociety 92, 6654 (1970) and 92, 6646 (1979)] from the correspondingcarboxylic acids, were ortho-metallated and reacted withdimethylformamide. Acid hydrolysis of the product from this reactionfollowed by standard isolation as detailed in Synthesis Examples 2 and 3furnished the required hydroxy phthalides (VIb) and (VIc). ##STR7## Thecyanophthalides (IIIa-c) were then prepared by reaction with alkalimetal cyanide, acidification, and then cyclization with Vielsmeierreagent by a procedure similar to that described in U.S. patentapplication Ser. No. 692,584, filed on Jan. 18, 1985. Details arepresented in Examples 1-3. ##STR8##

Quinone monoketals useful in the present invention are available fromthallium oxidation of p-methoxyphenols or anodic oxidation of1,4-dimethoxybenzenes followed by mild acid hydrolysis. They are bestobtained by anodic oxidation of 1,4-dimethoxy aromatic systems followedby monohydrolysis of the quinone bisketal.

For a discussion of tne synthesis of quinone monoketals, reference maybe made to Swenton, John S., "Anthracycline Antibiotics," H. El Khadem,Ed.; Academic Press, Inc., New York, 1982; Dolson et al., supra; Chenardet al., "Annelation Reaction of Quinone Monoketals Studies Directed atan Efficient Synthesis of Anthracyclinones," J. Org. Chem. 49, 318-325(1984); and J. S. Swenton, Acc. Chem. Res. 16, 74-81 (1983).

Much of the work which has been done using annelation reactions has beendone using quinone monoketals in which the oxygen functions at theeventual C-7 and C-9 positions have a trans relationship. Epimerizationof the C-7 oxygen and separation of the isomers are required to obtainthe active antibiotic. The separations of anthracyclinones such as (±)4-demethoxydaunomycinone and (±) daunomycinone from their epi-isomers isparticularly difficult. Consequently, in the preferred synthesis thecorrect A-ring stereochemistry is established early, before thetetracyclic structure is formed.

In accordance with the preferred embodiments of the present invention,the quinone monoketal is synthesized with the C-7 and C-9 oxygens incis-relationship. This can be accomplished using the reaction sequencetaught by Swenton, "Anthracycline Antiobiotics" p. 189 or by thefollowing route: ##STR9##

The basic bicyclic system 11 is prepared by the general approach of Wongand Schwenk, Can J. Chem., 49, 2712 (1979). These procedures, detailedin the examples below, allow preparation of 10 in 63% overall yield withno purifications except a simple recrystallization in the final step.The liquid hydrogen fluoride cyclization of the anhydride 10 to thetetralone 11, while useful in small-scale work, is impractical forlarge-scale synthesis. Although a number of other Friedel-Craftscatalysts do not give good yields in the 10 to 11 conversion, titaniumtetrachloride in methylene chloride effects the ring closurereproducibly on a 100 gram scale in 90% yield.

The two CO groups of 11 are differentiated chemically by esterificationfollowed by thioketalization to give 12. Several attempts were made tointroduce the hydroxyl group alpha to the ester function in 12.Oxygenation of the ester enolate of 12 proved difficult. Oxygenationwith oxodiperoxymolybdenum (pyridine) (hexamethylphosphoric triamide),the MoO₅ HMPA reagent, gives a complex mixture of products. Apparently,oxidation of the thioketal competes with enolate oxidation. Oxygenationof the ester enolate with oxygen in the presence of triethylphosphitegives primarily recovered starting material.

The preferred method involves conversion of the ester to the ketone viathe Corey procedure (J. Am. Chem. Soc., 87 1345 (1965). This conversionwas initially conducted on the methyl ester of 12; however, the yieldsof tne reaction were quite variable, and poor yields were obtained onlarger scale (50 g) reactions. Surmising that part of the difficultycomes from the high insolubility of the methyl ester in the solventsystem, the reaction was then performed on the lower-melting and moresoluble ethyl ester. Using the ethyl ester 12, the conversion to 13 wasconducted routinely on a 100-gram scale in over 90% yield. The requiredtertiary hydroxyl group is then introduced via oxygenation of the ketoneenolate. Tne yields for this oxygenation were only reproducible whenfreshly distilled dimethylformamide was employed as solvent and theoxygen uptake monitored. Otherwise, an induction period was often noted,and, unless the reaction was quenched at the proper time, low yields of14 resulted. The results of this oxygenation are surprising. It mighthave been anticipated that the reaction would fail since the sulfur ofthe thioketal could be easily oxidized oy the hydroperoxide intermediateformed in the reaction.

The racemate 14 can be resolved using S(-)-α-methylbenzylamine. Sinceboth antipodes of tne amine are commercially available, a successfulresolution affords the molecule of natural absolute configuration.Reaction of 14 with S(-)-α-methylbenzylamine followed byrecrystallization from ethyl acetate affords a 25% yield of opticallypure imine which is hydrolyzed to a levorotatory hydroxy ketone, (-)-14.While the yield of the resolution is only 25%, the enriched iminerecovered from the initial resolution can be hydrolyzed, and tneenriched (R)-14 reacted with R(+)-α-methylbenzylamine to afford afterresolution and hydrolysis pure (R)-14. The mother liquors from thissequence of reactions are then resolved with (-)-α-methylbenzylamine toafford additional (S)-14. In practice, two such cycles gave a 35% yieldof (S)-14 from racemic 14. Conversion of (-)-14 to natural daunomycinone(vide supra) establishes the configuration of 14 as the desired naturalconfiguration.

Ketalization of 14 under standard azeotropic conditions sometimes leadsto decomposition products as well as the desired ketal, but mildketalization conditions give the ketal 15 in quantitative yield. Variousmethods for reduction of 16, available from thioketal hydrolysis of 15,afford a difficultly separable cis/trans mixture of diols. However,potassium tri-sec-butylborohydride (K-selectride) reduction of 16affords almost exclusively the required cis-diol 17.

Previous studies of the annelation reaction with the phenylsulfonylanion indicated that it was necessary to protect the tertiary OH groupto obtain modest yields of tetracyclic product. Since the benzylic OHgroup of 17 needs to be protected for the anodic oxidation step leadingto the quinone bisketal, various methods for protecting both OH groupsin 17 were examined. While the benzylic hydroxyl group is easilyfunctionalized, functionalization of both it and the tertiary OH groupof 17 could not be performed cleanly with either t-butyldimethylsilylchloride or chloromethylmethyl etner. Apparently, the steric bulk of theethylene glycol ketal and the other cis oxygen substitutent hindersfunctionalization of the tertiary position. The difficulty in protectingboth hydroxyl groups led to the examination of the chemistry of themonoblocked system 18.

Anodic oxidation of 18 in a single cell produces in excellent yield therespective quinone bisketal, 19, which is directly hydrolyzed to amixture of two regioisomeric monoketals in a ratio of ca 83:17 asdetermined by HPLC analysis. The major isomer, which could oe obtainedin a pure form by recrystallization, was assigned as 20 by analogy tothe directing effect of an allylic oxygen substitutent on model quinonebisketal hydrolyses. This assignment was subsequently confirmed by theuse of the monoketal in the synthesis of (+)-daunomycinone.

The quinone monoketal is then reacted with the cyanophthalide anion asfollows: ##STR10##

The annelation reaction is typically conducted at 0° C. The highestyields have been obtained using a homogeneous medium of tetrahydrofuranand DMSO as the solvent and dimsyl anion as base. Those skilled in theart will appreciate that other solvent and base systems can be used withvarying results. Potentially useful solvents include ethers (especially1,2-dimethoxyethane), alcohols (especially t-butyl alcohol),dimethylformamide, and hexamethylphosphoramide. Potentially useful basesinclude alkali and ammonium hydroxides (especially under phase transferconditions), metal alkoxides [especially alkali, cesium (III) salts, andt-butyl alkoxides], and alkali metal salts of hindered amines.

A proposed laboratory tecnnique for reacting the cyanophthalide and thequinone monoketal is to add methyl lithium dropwise to a 0° solution ofDMSO and THF (V/V=52:130). The cyanophthalide in DMSO is then added overabout 1 minute. After stirring for 5 minutes, the solution is cooled toabout -40°, and the monoketal 20 in THF is added rapidly. The coolingbath is removed and the mixture is stirred for several hours at roomtemperature.

Hydrolysis of the ketal and demethylation of the aromatic methoxyl groupgives the anthracyclinone an excellent overall yield.

Anthracycline aglycones prepared in accordance with the presentinvention can be coupled with a sugar such as daunosamine according tothe teachings of Acton et al, supra, to produce the antibiotic or usingother coupling reactions known in the art. Tne reaction of theanthracyclinone with a 1'-chlorosugar in a Koenigs-Knorr reaction isoften used. See also U.S. Pat. No. 4,067,969.

While tne synthesis has been illustrated for daunomycinones, it can alsobe used to prepare the adriamycinones. The anthracyclinones availablefrom this chemistry can be functionalized at the C-14 methyl group asoutlined by Arcamore et al. in U.S. Pat. Nos. 4,046,878, 4,039,663 and4,325,947. Typically, this involves bromination at C-14 followed bytreatment witn aqueous sodium formate.

2'-Halo derivatives are preferably prepared from1,6-anhydro-3,4-di-O-acetyl-2,6-dideoxy-hex-1-enitols sucn as3,4-di-O-acetyl-L-fucal or 3,4-di-O-acetyl-L-rhamnal. These sugars canbe prepared as described in B. Iselin and T. Reichstein, Helv. Chim.Acta, 27, 1146, 1200 (1944).

The aglycon is usually reacted with 3,4-di-O-acetyl-L-rhamnal or3,4,-di-O-acetyl-L-fucal in an anhydrous mixture of acetonitrile andtetrahydrofuran followed by the addition of a halogenating agent such asN-iodosuccinimide or N-bromosuccinimide. The halogenating agent isgenerally used in a stoichiometric excess, e.g., 1.5 to 3 times theamount of the aglycon on a molar basis. This syntnesis is described inU.S. Pat. No. 4,427,664 to Horton et al. 4'-Halo derivatives can beprepared in a manner analogous to that described in European Pat.Specification No. 81107618.1 and U.K. Application No. 2,118,540published Nov. 2, 1983.

The invention is illustrated in more detail by the followingnon-limiting examples. Unless otherwise indicated, all temperatures aregiven in degrees Celsius.

SYNTHESIS EXAMPLE 1 a. 3-Hydroxy-7-fluoro-1(3H)-isobenzofuranone

To a -70° C. solution of the dimethylacetal of 3-fluorobenzaldehyde (5.3g, 31 mmol) in THF (35 ml) was added dropwise sec-butyllithium (22.2 mlof a 1.4 M solution in hexane), and the red solution was stirred for 0.5h. The solution was saturated with CO₂ for 5 min with tne red colordissipating to yield a light yellow solution. After 10 min the reactionwas allowed to warm to room temperature, and after 30 min HCl (3.5 ml)was added. After concentration the solution was made basic with 5% KOH(25 ml), the neutral material extracted with Et₂ O (2×40 ml), the baselayer acidified to pH 1 witn HCl, and the product extracted with EtOAc(2×75 ml). Work up as usual afforded 4.5 g (86%) of the title compound,mp 11°-119°, suitable for use in the next step. Recrystallization ofthis material from EtOAc/PE gave analytically pure material: mp126°-127° C.; IR 3400 (br s), 1740-1760 (structured s), 1630 (m), 1485(m), 1300 (m), 1085 (s), 910 (m), 760 (m); ¹ H NMR 7.8-7.1 (highlystructured m, 3 H), 6.60 (br s, 1H); exact mass calcd for C₈ H₅ O₃ F m/e168.0227, obsd 168.0226.

b. 3-Cyano-7-fluoro-1(3H)-isobenzofuranone

A solution of KCN (2.1 g, 30.8 mmol), water (3.5 ml), and the productsof part a (0.71 g, 4.2 mmol) was cooled to 0° C. Concentrated HCl (14ml) was added, and the solution was stirred for 10 min and thenextracted with EtOAc (3×50 ml). Workup as usual gave a yellow-orange oilwhich was dried under vacuum, dissolved in CH₃ CN (8 ml), and added tothe Vilsmeier salt prepared from oxalyl chloride (1.3 g) and DMF (1.23ml) in the usual manner. After stirring for 1 min at 0° C., pyridine(1.7 ml) was added, and the solution was stirred for an additional 15min. Workup as for the parent system gave a dark red oil which wasrecrystallized from CH₃ OH to afford 0.425 g (57% of the title compoundin two crops, mp 117°-120° C. An additional crystallization (CH₃ OH)gave the pure material: mp 121° -123° C.; IR 1780 (s), 1630 (m), 1610(m), 1490 (m), 1280 (m), 1260 (m), 1070 (m), 1025 (m), 995 (s), 800 (m);¹ H NMR 8.0-7.6 (str m, 1 H), 7.6-7.2 (str m, 2 H), 6.1 (s, 1 H); exactmass calcd for C₉ H₄ NO₂ F m/e 177.0227, obsd 177.0233.

SYNTHESIS EXAMPLE 2 a. 4-Fluoro-3-hydroxy-1(3H)-isobenzofuranone

To a -78° C. solution of the oxazoline derivative (7.8 g, 40.4 mmol),prepared from 3-fluorobenzoic acid in tne usual manner, in THF (50 mL)was added over 15 min sec-BuLi (29 mL of 1.4 M solution). After 30 min,DMF (6.3 mL) was added, and the reaction mixture was stirred at -78° C.for 2 hr. Tne reaction was then quenched with water (10 mL), and themixture was acidified witn HCl and allowed to stir for 12 hr. The layerswere separated, the aqueous layer was extracted with EtOAc, and thecombined organic layer was concentrated in vacuo. Tnis material wasdissolved in 5% KOH (50 mL), and the base layer was washed with Et₂ O(2×100 mL) to remove neutrals. The resulting base layer was added to 0°C. solution of concentrated HCl (25 mL), and the resulting cold acidicsolution was extracted with EtOAc (3×75 mL). Workup and drying gave atacky yellow solid (4.4 g) which was recrystallized from CHCl₃ to givethe title compound (1.97 g, 30%), mp 97°-100° C.

b. 4-Fluoro-3-cyano-1(3 H)-isobenzofuranone

The above hydroxyphthalide (0.5 g, 3.0 mmol) and KCN (1.5 g) weredissolved in water (5 mL) and cooled to 0° C. A little ice was addedfollowed by dropwise addition of concentrated HCl (10 mL), whereupon thesolution turned cloudy. The mixture was stirred for 5 min and thenextracted with EtOAc (2×75 mL). Workup and drying gave a thick yellowoil which was used directly in the next step. To the Vielsmeier saltprepared in the usual way from CH₃ CN (7 mL), oxalyl chloride (0.92 g),and DMF (0.86 mL) was added the above material in CH₃ CN (6 mL). Afterstirring for 1 min, pyridine (1.2 mL) was added, and the mixture wasstirred an additional 15 min. The reaction mixture was then poured into5% HCl (75 mL). Extractive workup (EtOAc) and concentration gave anorange/yellow solid which was chromatographed on silica gel (CH₂ Cl₂ aseluant) to give the title compound (0.36 g, 60%) from hydroxyphthalideas light yellow crystals, mp 93°-96° C.

SYNTHESIS EXAMPLE 3 a. 4,7-Difluoro-3-hydroxy-1(3H)-isobenzofuranone

To a -78° C. solution of the oxazoline derivative (7.95 g, 37.7 mmol),prepared from 2,5-difluorobenzoic acid in the usual manner, in THF (50mL) was added over 15 min sec-BuLi (29 mL of 1.3 M solution). After 30min, DMF (6.0 mL) was added, and the reaction was stirred at -78° C. for2 hr. The reaction was tnen quenched with concentrated HCl (5 mL). Tnesolvents were removed in vacuo, and the residue was taken up in 5 N HCl(5 mL) and heated on the steam bath overnight. After cooling to roomtemperature, the reaction mixture was extracted with EtOAc (3×200 mL).Standard workup gave a light brown solid (6.5 g, 93%, mp 125°-128° C.).

b. 4,7-Difluoro-3-cyano-1(3H)-isobenzofuranone

The above hydroxy phthalide (3 g, 16.1 mmol) and NaCN (7.5 g) weredissolved in water (15 mL) and cooled to 0° C. Ice (2 g) was added,followed by dropwise addition of concentrated HCl (60 mL). The mixturewas stirred for 5 min and then extracted with EtOAc (3×50 mL). Workupand drying gave a thick yellow oil which was used directly in tne nextstep. To the Vielsmeier salt prepared in the usual way from CH₃ CN (39mL), oxalyl chloride (3.4 mL), and DMF (4.6 mL), was added the abovematerial in CH₃ CN (30 mL). After stirring for 1 min, pyridine (7.2 mL)was added, and the mixture was stirred an additional 15 min. Thereaction mixture was then poured into 5% HCl (200 mL). Extractive workup(EtOAc, 3×75 mL) and concentration gave an orange/yellow solid which waschromatographed on silica gel (CH₂ Cl₂ as eluant) to give the titlecompound (1.5 g, 48%) from hydroxy phthalide as light yellow crystals,mp 87°-89° C.

SYNTHESIS EXAMPLE 4 Dimethyl-(2,5-dimethoxybenzylidine) malonate

A mixture of 2,5-dimethoxybenazldehyde (300 g, 1.8 mol), dimethymalonate(240 ml, 262 g, 1.98 mol), piperidine (9.0 ml), HOAc (3 ml), and benzene(300 ml) was heated to reflux in an apparatus equipped with a Dean-Starktrap (34 ml of lower phase was collected over 14 hr). The mixture wasdiluted with an equal volume of Et₂ O and washed with 00 ml portions of5% HCl, 5% NaHCO₃, and brine. Concentration in vacuo yielded a yellowoil which was diluted with Et₂ O/H and cooled to givedimethyl-(2,5-dimethoxybenzylidene) malonate (471 g, 93%) as yellowcrystals suitable for use in the next step. A sample recrystallized fromEt₂ O showed: mp 59°-60°; IR 2950 (m), 1740 (s,) 1725 (s), 1620 (m),1500-1400 (m, structured), 1272-1150 (s, structured); ¹ H-NMR (60 MHz,CCl₄) 7.80 (s, 1H), 6.78 (m, 3H), 3.75 (overalapping s, 6H), 3.68 (s,3H), 3.60 (s, 3H); exact mass calcd for C₁₄ H₁₆ O₆ m/e 280.0947, obsd280.0936.

SYNTHESIS EXAMPLE 5Trimethyl-3-(2,5-dimethoxyphenyl)-1,2,2,-propanetricarboxylate

A mixture of the product from Synthesis Example 4 (100 g, 0.36 mol) and10% Pt-C (2.0 g) in THF (225 ml) was hydrogenated in a Paar apparatus(initial pressure 59 lb/in.², final pressure 27 lb/in.²) for 5 hr. Thesolution was filtered througn Celite, and tne colorless filtrate wasused directly in the next step. Distillation of a portion of thematerial through a shortpath head afforded 90% of a colorless viscousoil: bp 172°-174°/0.6 mm; IR (neat) 2970 (s), 1740 (s), 1500 (s), 1440(s), 1280 (s), 1230 (s), 1160 (s), 1010 (s), ¹ H-NMR (60 MHz, CCl₄) 6.60(m, 3H), 3.80 (s, 3H), 3.70 (s, 3H), 3.65 (s, 6H), 3.58 (obscured, 1H),3.10 (d, J=7.5 Hz, 2H).

The hydrogenation solution was diluted with sufficient THF to make thetotal volume about 1 liter and then placed in a 2-1, 3-necked flaskunder N₂ while NaH (17 g, 60% by weight) in mineral oil was added over 1hr. After H₂ evolution ceased, methyl bromoacetate (59.8 g) was addedover 15 min. The solution was heated to reflux for 5 hr and then the THFwas removed in vacuo to afford a white oily solid (a mixture of productNaBr, mineral oil, and unknown impurities). Tnis material was useddirectly in the next step. Standard workup of a portion of the materialfollowed by recrystallization of the product from Et₂ O/PE gave thetriester as a white crystalline solid: mp 102.5°-104.0°; IR 1730 (s),1495 (s), 1426 (s), 1310 (s), 1280 (s), 1220 (s), 1190 (s), 1040 (s); ¹H-NMR (60 MHz, CCl₄), 6.60 (brd, 2H), 6.42 (brd, 1 H), 3.55-3.70(overlappings, 15H), 3.32 (s, 2H), 2.70 (s, 2H); exact mass calc for C₁₇H₂₂ O₈ m/e 354.1315, obsd 354.1305.

SYNTHESIS EXAMPLE 6 (2,5-Dimetnoxybenzyl) succinic anhydride 10

The crude triester from two runs as described in Synthesis Example 5[200 g of starting dimethyl-(2,5-dimetnoxybenzylidine) malonate] wasdissolved in hot EtOH (600 ml), water (1200 ml) and KOH (360 g) wereadded, and the homogeneous solution was heated to a gentle reflux for 14hr. The resulting orange solution was cooled and then extracted withCHCl₁₃ (2×400 ml). Tne light orange aqueous phase was cooled in ice andslowly acidified with conc HCl (600 ml). After 5 hr at 0°, the solid wascollected and dried to constant weight under vacuum to afford 182.2 g(81% overall from Knoevenagel product) of white powdery solid.

Tne crude triacid (182.2 g, 0.584 mol) was added to Ac₂ O (1.0 1), andthe solution was slowly heated to reflux. The solid gradually dissolved,and CO₂ evolution was apparent. After heating for 2 hr, the solution wasconcentrated by distillation at atmospheric pressure (500 ml ofdistillate was collected). The resulting cloudy solution was filtered(3.4 g of white solid, probably NaBr, was collected), and the remainingAc₂ O was removed in vacuo at about 80°. The dark brown oil was thenpoured into a 1 L flask, and the flask was rinsed, using a minimumamount of CHCl₃. The solution was rapidly swirled while hexane (about350 ml) was added, and then tne product was allowed to crystallize.After collection of the solid and drying, white anhydride (122 g, 84%from the crude acid) was obtained: mp 74°-76° (lit. mp 75°-76°); IR 1860(s), 1780 (s), 1500 (s), 1220 (s), 1170 (s), 1140 (s), 928 (s), 915 (s);¹ H-NMR 6.69 (2s, 3H), 3.75 (s, 3H), 3.73 (s, 3H), 3.20-2.74 (m, 5H).

SYNTHESIS EXAMPLE 7 1,2,3,4-Tetrahydro-5,8-dimethoxy-4-oxo-2-naphthoicacid 11

The anhydride (106 g, 0.42 mol) in CH₂ Cl₂ (500 ml) was added to astirred solution of TiCl₄ (110.2 ml, 190.6 g, 1.0 mole) in CH₂ Cl₂ (1600ml) at room temperature over a period of 20 min. A slight warming of themixture was noted, and the solution was then stirred at room temperaturefor 1 hr. The mixture was then cooled in ice, and the CH₂ Cl₂ layer waspoured onto ice (300 g). Some product precipitated and was collected;however, tne majority of material remained as a reddish oily solid onthe walls of the reaction flasx. Water (850 ml) was added to tne flask,and the solution was vigorously stirred until the reddish-brown materialwas converted into a light yellow solid. Tnis material was filtered andcombined with the previous solid, washed with water and dried (first inthe air and then under vacuum) to ootain the keto acid (95.4 g. 90%), mp199°-201° (lit. mp 207°-208° ), suitable for use directly in the nextstep; IR 1740 (s), 1680 (s), 1475 (s), 1280 (s), 1270 (s), 1260 (s),1170 (s), 1080 (s), ¹ H-NMR 6.90 (ABq, Δv=15.6 Hz, J=9 Hz, 2H), 3.85 (s,3H), 3.82 (s, 3H), 3.6-2.7 (m, 5H).

SYNTHESIS EXAMPLE 8Ethyl-1,2,3,4-tetrahydro-5,8-dimethoxy-4-oxo-2-naphthoate Cyclic4-(Ethylene mercaptole), 12

A mixture of the keto acid from Synthesis Example 7 (142 g 0.57 mol),abs EtOH (100 ml), benzene (550 ml), and p-TsOH (0.5 g) was heated toreflux under a 39 cm Vigreux column and a Dean-Stark trap. Tne pottemperature was adjusted so that the benzene/EtOH/H₂ O azeotropedistilled, and the reaction was allowed to proceed for 24 hr.Conventional workup afforded 95% of tne ester as a light tan solid.Recrystallization of this material from EtOH afforded the ethyl ester asa white solid: mp 96°-98°; IR 1730 (s), 1680 (s), 1585 (s), 1475 (s),1255 (br s), 1085 (s); ¹ H-NMR (60 MHz) 6.88 (AB q, Δv=15 Hz, J=9 Hz,2H), 2.23 (l, J=6 Hz, 2H), 3.85 (s, 3H), 3.82 (s, 3H), 3.39-2.82 (m, 5H)1.23 (t, J=6.5 Hz, 3H); exact mass calc for C₁₅ H₁₈ O₅ m/e 278.1154,obsd 278.1162.

More conveniently, the solvent from the crude esterification mixture wasdistilled at atmospheric pressure, and the solid residue was dried undervacuum for several hours. To this mixture were added benzene (1.51),1,2-ethanedithiol (0.432 mol) and BF₃ Et₂ O (0.5 ml). This mixture washeated to reflux under a Dean-Stark trap for 12 hr, and then the solventwas distilled at atmospheric pressure until the volume of the reactionmixture was about 150 ml. The cooled mixture was then diluted with anequal volume of PE and cooled in ice. The title compound, obtained as atan solid (184 g, 90%, mp 107°-109°), was used directly in the nextstep. A sample recrystallized from benzene/H gave white crystallinematerial; mp 114°-116°; IR 2950 (m), 2930 (m), 1725 (s), 1590 (m), 1475(s), 1460 (s), 1285 (s), 1255 (s), 1065 (s); ¹ H-NMR (90 MHz) 6.75 (s,2H), 4.21 (q, J=7.6 Hz, 2H) 2.86 (s, 3H), 3.76 (s, 3H), 3.57-3.42 (m,4H), 3.2-2.3 (m, 5H), 1.31 (t, J=7.5 Hz, 3H); exact mass calc for C₁₇H₂₂ O₄ S₂ m/e 354.0960, obsd 354.0970.

SYNTHESIS EXAMPLE 91,2,3,4-Tetrahydro-5,8-dimethoxy-4-oxo-2acetylnaphthalene, 13

To a 3-1, round bottomed flask equipped with a stirrer and condenser andmaintained under N₂ was added NaH (37.8 g of a 60% oil dispersion, 0.95mol). The NaH was washed with PE (2×25 ml), and DMSO (400 ml) was added.The material was slowly heated to 65°-70° and maintained at thistemperature until H₂ evolution ceased (2 hr). The mixture was cooled to0°, and THF (400 ml) was added. The crude product from the previous step(134 g, 0.38 mol) was dissolved in THF (535 ml) and added to the rapidlystirred solution. The ice bath was removed, and the brownish-redsolution was stirred for 1 hr and then poured into water (4 1). Thismixture was carefully acidified to pH 4 by dropwise addition of concHCl, and tne solution was extracted with CH₂ Cl₂ (4×750 ml). Theextracts were combined and washed with water (2×2500 ml), tne bulk oftne solvent was removed oy distillation at atmospheric pressure, and theremaining volatiles were removed from the thick yellow oil by vacuumdrying for 14 hr. The crude ketosulfoxide showed: ¹ H-NMR 6.78 (s, 2H),4.00 (s, 2H), 3.89 (s, 3H), 3.82 (s, 3H), 3.51-3.39 (m, 4H), 3.20-3.00), (m,5H), 2.71 (s, 3H).

The material was dissolved in THF and divided into two batches for theAl/Hg reduction. Aluminum foil (57 g, 1-in. squares) was placed in a5-1, 3-necked, round-bottomed flask equipped with an efficient condenserand an overhead stirrer, and 2% aqueous HgCl₂ (1.01) was added. THesolution was swirled for 30 sec, then the HgCl₂ solution was poured off,and a solution of the compound in THF (2400 ml) was added, followed byaddition of water (250 ml).

The Al is very reactive after amalgamation. Consequently, these stepsmust be done rapidly since a fire could result. The reaction must bemonitored closely since it becomes sufficiently exothermic that coolingmust be employed; however, the reaction temp should oe maintained above50°. After 2 hr, the H₂ evolution ceased, and the mixture was filteredthrough Celite. Tne THF was removed in vacuo and then used to wash thealuminum salts; this process was repeated three times. The productsolidified upon concentration to afford 98 g (80%) of light yellowsolid, mp 124°-129°, which was used directly in the next step. The yieldof product varied from 80% to as high as 95% on 50 gram runs. A samplerecrystallized from EtOAC/H showed: mp 128°-130°; IR 1710 (br s), 1600(m), 1475 (br s) 1435 (s), 1280 (s), 1255 (s), 1090 (s); ¹ H-NMR (90MHz) 6.75 (s, 2H), 3.38 (s, 3H), 3.74 (s, 3H), 3.6-3.39 (m, 4H),3.1-2.45 (m, 5H). 2.26 (s, 3H); exact mass calc for C₁₆ H₂₀ O₃ S₂ m/e324.0854, obsd 324.0861.

SYNTHESIS EXAMPLE 101,2,3,4-Tetrahydro-5,8-dimethoxy-4-oxo-2-acetyl-2hydroxy naphthalene, 14

To a stirred -24° C. solution of the ketone (8.9 g, 27.4 mmol), t-BuOH(23 ml), freshly distilled DMF (71 ml), and (EtO)₃ P (5 ml, 27.4 mmol)maintained under N₂ was added a -50° solution of t-BuOH (6.2 g, 55.2mmol) in DMF 26 ml O₂ was introduced into the system and after 15 minone equivalent of O₂ (670 ml) was absorbed. The light orange mixture wasthen quenched by addition of HOAc (5 ml) affording a light yellowmixture. Two such runs were combined and concentrated at 60°/0.2 mm andthe residue worked up as usual to afford a light yellow syrup. Themajority of remaining volatiles were removed at room temp/10⁻³ mm over24 hr. Trituration of the resulting semi-solid with Et₂ O/EtOAc affordedin two crops 12.2 g(65%) of light yellow solid, mp 143°-147° suitablefor use in the next step. Recrystallization of this material from EtOAcgave in three crops 10.6 g (56%) of crystalline hydroxy ketone, mp148°-150°. The analytically pure material showed mp 151°-153° (lit. mp152.5°-153.0°).

Resolution of (+)-14

A mixture of (±)-14 (4.8 g, 0.014 mol), benzene (100 ml), Linde 4Åsieves (10 g), and (+)-α-methylbenzylamine (3.6 ml, 0.028 mol) washeated reflux overnight in an apparatus equipped with a Dean-Stark trap.The mixture was then filtered through a Celite pad and concentrated invacuo to afford, after trituration with Et₂ O, the diastereomeric iminesas an off-white powder, mp 165°-178°. Tne ratio of the diastereomericimines can be determined by integration of the Me groups at about 1.35in the 200 MHz ¹ H-NMR spectrum. In a typical run this crude product wasdissolved in boiling EtOAc (40 ml), and then the volume was reduced to25 ml. Slow cooling to room temperature gave clear crystals which werefiltered, washed with EtOAc (10 ml) and Et₂ O (15 ml), and dried toyield 1.55 g (25%) of colorless needles: mp 195-95.5; [α]_(D) ²⁰(dioxane)=-29.5°; IR (K Br) 3240 (m), 1660 (s), 1475 (s), 1460 (s), 1435(s), 1260 (s), 1250 (s), 1080 (s); ¹ H-NMR (200 MHz, C₆ D₆) 7.25-7.00(m, 5H), 6.51 (AB a, J=4.4 Hz, Δv=12.9 Hz, 2H) 4.37 (q, J=6 Hz, 1H),3.55 (s, 3H), 3.4-3.3 (m, 2H), 3.29 (s, 3H), 3.17-2.75 (m, 7H), 1.42 (s,3H), 1.25 (d, J=6 Hz, 3H); exact mass calc for C₂₄ H₂₉ NO₃ S₂ m/e443.1588, obsd 443.1600.

The combined mother liquors were concentrated in vacuo and thenhydrolyzed to enriched (s)-14 by adding THF (30 ml) and 5% aqueous HCl(7 ml). Tnis solution was stirred at room temp for 0.5 hr, and then theTHF was removed in vacuo. The aqueous layer was extracted with CH₂ Cl₂(3×30 ml), and the combined organic phases were worked up as usual toafford an oil which crystallized on standing. This hydroxy ketoneenriched in (S)-14 was reacted as described above except with(-)-α-methylbenzylamine to yield 1.81 g (29%) of imine: mp 195-195.5%;[α]_(D) ²⁰ (dioxane)=+29.5°. Note that this latter imine is the compoundhaving the natural configuration of the anthracyclinone A-ring.

A solution of 5% aqueous HCl (25 ml) was added to a stirred solution ofthe imine above (3.05 g, 8.9 mmol) in THF (110 ml). The resulting lightyellow solution was stirred for 10 min at room temperature and thenconcentrated in vacuo to remove the majority of the THF. The residue wasextracted with CH₂ Cl₂ (3×30 ml) and worked up as usual to afford thelevorotatory hydroxy ketone, (s)-14. Recrystallization of this materialfrom EtOAc gave 2.05 g (89%) of white product: mp 178.5°-179.5°; [α]_(D)²⁰ (CHCl₃)=24.4° [lit. mp 178.5°-179.5°, [α]_(D) ²⁰ (CHCl₃ =-24.4°].

SYNTHESIS EXAMPLE 111,2,3,4-Tetrahydro-5,8-dimethoxy-4-oxo-2-acetyl-2-hydroxynapthalene.Cyclic 4-(ethylene mercaptole), cyclic 2-ethylene-glycol ketal, 15.

A mixture of ketone 14 (61.7 g, 0.18 mol), distilled ethylene glycol(312 ml), commercial trimethylorthoformate (240 ml), and anhydrousp-TsOH (1 g) was combined in the above order and stirred in an oil bathat 37°-40° for 24 hr. The heterogeneous solution (white solid in a blueliquid) was poured into 20% KOH (1200 ml) and stirred for 1.5 hr in anice bath. The solution was then diluted with water (2 l), stirred for anadditional hour, and then filtered. Drying the white solid overnight invacuo gave the ketal as a cream-colored solid (69 g, 100%), mp 163°-166°(mp 162.4°-163°).

For (S)-15 the homogeneous mixture was poured into 20% CH₃ OH/KOH, andafter 30 min, ice was added to precipitate the crude solid. Theoptically pure material showed: mp 130°-131° [α]_(D) ²⁰ (CHCl₃ -38.0°,[lit. mp 144°-146°, [α]_(D) ²⁰ (CHCl₃)=-42.4°].

SYNTHESIS EXAMPLE 12 Compound 17

The thioketal 15 (22.0 g, 57.3 mmol) was dissolved in CH₃ OH (3200 ml)and water (400 ml), and to the vigorously stirred mixture were addedHgCl₂ (22 g, 81 mmol) and yellow HgO (16.6 g, 77 mmol). The progress ofthe reaction was monitored oy TLC (Et₂ O, phosphomolybdic acid spray,starting material is blue witn R_(f) 0.4 and product is much lessintense with R_(f) 0.1), and the reaction was judged complete after 20min. Tne solution was filtered tnrough Celite and concentrated at about50°-55° in vacuo until the volume was about 500 ml. The filteredHg-salts were slurried with CH₂ CL₂ (3×300 ml) and this solvent was usedfor extraction of tne product from the concentrated aqueous solution.Workup gave 15.4 g (88%) of the β-hydroxy ketone as a light yellow solidwhich was used directly in the next step, mp 173°-176° (lit. mp177.5°-178.0).

The optically pure material showed: mp 182°-183°, [α]_(D) ²⁰(CHCl₃)=+13.9° [lit. mp 182.5°-184.0°, [α]_(D) ²⁰ (CHCl₃ =+14.0°].

A slurry of the crude hydroxy ketone (15.4 g, 0.05 mol) from above inTHF (500 ml) was cooled in dry ice for about 0.5 hr. and tnen potassiumtri-sec-butylborohydride (100 ml of a 1M THF solution, 0.1 mol) wasadded dropwise over 0.5 hr. The solution was stirred for 1 hr at dry icetemperature and tnen for 2 hr at room temperature. The nomogeneousyellow solution was cooled in ice, and 20% KOH (225 ml) was addedcautiously followed oy dropwise addition of 30% H₂ O₂ (48.6 ml). Thesolution was stirred at room temperature for 1 hr, and then tne excessperoxide was destroyed by addition of sodium thiosulfate. Afterconcentration in vacuo, the aqueous solution was extracted with CH₂ Cl₂(3×200 ml). Workup and trituration with Et₂ O gave a light yellowcrystalline solid. This material was crystallized from CH₂ Cl₂ /Et₂ O togive 13.7 g (89%) of diol, mp 125°-126.5°.

Tne optically pure material showed: mp 141°-143°, [α]_(D) ²⁰(CHCl₃)=+5.3; IR 3530 (br s), 3430 (br s), 1495 (s), 1280 (s.sh), 1275(s), 1110 (s), 1095 (s), 1080 (s); ¹ H-NMR (200 MHz, D₂ O wash) 6.66 (s,2H), 5.14 (br, 1H), 4.03 (s, 4H), 3.81 (s, 3H). 3.75 (s, 3H), 3.08 (dd,J=17.7, 2.4 Hz, 1H), 2.65 (d, J=17.7 Hz 1H), 2.36 (d oft, J=2.4, 2.4,18.9 Hz, 1H), 1.92 (dd, J=4.9, 18.9 Hz, 1H), 1.42 (s. 3H); exact massfor C₁₆ H₂₂ O₆ calc m/e 310.1416, obsd 310.1423.

SYNTHESIS EXAMPLE 13 Compound 18.

A mixture of the diol 17 (20 g, 0.065 mol), imidazole (20 g, 0.29 mol),t-butyldimethylsilyl chloride (20 g, 0.13 mol) dissolved in dry DMF (200ml) was stirred at 45° for 24 hr, after which time TLC (ca 80% Et₂ O/H)showed no starting diol. The bulk of the DMF was removed at 45°/1 mm,and the residue was partitioned between CH₂ Cl₂ (45 ml) and sat. NaHCO₃(200 ml). Standard workup gave a thick oil which solidified to give theprotected diol (27 g, 96%) which was used directly in the next step. Theanalytical sample of the material was obtained by recrystallization fromCH₃ OH/H₂ O and showed: mp 106°-107°; IR 3440 (m), 1600 (m), 1480 (s),1465 (m), 1260 (s), 1074 (s), 1045 (m), 980 (m), 850 (m), 780 (m), ¹H-NMR (200 MHz) 6.7 (center of AB, J=8.7 Hz Δv=21.4 Hz 2H), 5.33(unresolved t, 1H), 5.29 (s, 1H), 4.03 (s, 4H), 3.76 (s, 6H), 3.12 (dd,J=2.16 Hz, 1H), 2.70 (d, J=16 Hz, 1H), 2.30 (d of poorly resolved t, J=14 Hz, 1H), 1.81 (dd, J=3.5, 14 Hz, 1H), 1.46 (s, 3H), 0.86 (s, 9H),0.25 (s, 3H), 0.08 (s, 3H); exact mass for C₂₂ H₃₆ O₆ Si calc m/e424.2281, obsd 424.2259.

The optically pure compound showed: mp 126°-127°, [α]_(D).sup.°(CHCl₃)=+7.9°.

SYNTHESIS EXAMPLE 14

Compound 19

A slurry of protected diol, 18, (14 g) and 1% KOH/CH₃ OH (600ml) wasanodically oxidized using the macro Pt electrode and the Pt sheet anodein a thermostated cell held at about 0° with a current of 1 amp. Thereaction was followed by UV analysis at 290 nm, and tne electrolysis wasterminated after the initial optical density decreased to 1% of itsinitial value (about 3 hr). The mixture was then neutralized with solidCO₂ and concentrated on the rotary evaporator. Concentration and dryingof the product gave a quantitative yield of bisketal which washydrolyzed directly to the monoketal. Recrystallization of a sample ofthe bisketal from Et₂ OH gave a white crystalline compound: mp 79°-81°.

The optically pure compound showed: m 103°-104° [α]_(D) ²⁰(CHCl₃)=18.7°; IR 3470 (s), 29560 (s), 2940 (s), 1110-1080 (vs, br),1032 (s), 1042 (s), 985 (s); ¹ H-NMR (CCl₄ CH₂ Cl₂) 6.06 (s, 2H), 4.73(distorted t, 1H), 4.58 (s, 1H). 3.87 (s, 4H), 3.12 (s, 3H), 3.09 (s,3H), 3.06 (s, 6H), 1.8-2.3 (m, 3H), 1.43 (dd, J=3.14 Hz, 1h), 1.23 (s,3H), 0.87 (s, 9H), 0.15 (s, 3H), 0.10 (s, 3H); ¹³ C-NMR 139.8 133.8132.5 131.5 111.8, 95.7, 95.2, 74.8, 65.3, 65.2, 63.9, 61.9, 51.2, 50.8,50.4 (3 C), 34.8, 31.7, 25.9, 18.5, 17.9, -4.8, -4.7.

A -20° C. solution of 8% HOAc/acetone (75 ml: 300 ml) was added to thecrude bisketal, and the homogeneous was stored at -20° C. for 48 hr. Thesolution was then poured into sat. NaHCO₃ (150 ml), and the majority ofthe acetone was removed on the rotary evaporator. Tne heterogeneoussuspension was extracted with CH₂ Cl₂ (2×250 ml), and the organic phasewas washed with sat. NaHCO₃ (150 ml). The solution was dried andconcentrated to give an amber oil which crystallized after being driedunder vacuum. The sample was dissolved in boiling Et₂ O (50 ml), and theEt₂ O was replaced with low-ooiling PE (50 ml). The material was allowedto crystallize at room temperature to afford 9.5 g (64%) of monoketal(20). TLC and 300 MHz ¹ H-NMR analysis showed that this was a 91:9mixture of regioisomers, mp 108°-110°, suitable for use in the couplingstep. A pure sample of monoketal was obtained by carefulrecrystallization from EtOAc/H:mp 117°-119°; IR 3480 (m), 1660 (s), 1090(s), 1060 (s), 1045 (s), 1045 (s); ¹ H-NMR (80 MHz) 6.57 (ABq,J=10 Hz,=27.5 Hz, 2H), 5.07 (distorted t, J 2.6 Hz, overlappings 2H), 4.00 (s,4H), 3.23 (s, 6H), 2.55 (ABq, Δv=31 Hz, J=19 Hz with lower field doublethaving J=1 Hz, 2H), 2.17 (d of distorted t, J=19 Hz, 1H), 1.67 (dd, J=3,19 Hz, 1H), 1.41 (s, 3H), 0.84 (s, 9H), 0.26 (s, 3H), 0.13 (s, 3H); ¹³C-NMR [(CD₃)₂ CO] 183.7, 153.0, 144.5, 135.8, 132.3, 112.4, 95.5, 76.6(2 C), 66.0, 64.0, 50.8, 34.6, 33.4, 26.1 (3 C), 18.9, 18.5, -4.6, -4.9(1 C missing). (Found:C, 59.82; H, 8.21, Calc for C₂₂ H₃₆ O₇ : C, 60.00;H, 8.18%)

SYNTHESIS EXAMPLE 15 (±)4-Fluoro-4-demethoxydaunomycinone

To a 0° C. solution of DMSO (35 mL)and THF (34 mL0 was added CH₃ Li (8.9mL of a 1.29 M solution). After 5 min,7-fluoro-3-cyano-1(3H)-isobenzofuranone (2.04 g, 11.5 mmol) in DMSO (34mL) was added, giving a golden brown solution. After an additional 5min, tne monoketal (3.9 g, 8.86 mmol) in THF (34 mL) was added,whereupon the solution turned deep red. The reaction mixture was allowedto stir at room temperature for 2 hr, and then the reaction was quenchedby the addition of 5% HCl (100 mL). The majority of tne THF was removedin vacuo, (CH₃)₂ CO (250 mL) was added, and the solution was allowed tostir for 12 hr. TLC analysis indicated that the t-butyldimethylsilylgroup was not completely removed, CHCl₃ was added to render the solutionhomogeneous, and the reaction mixture was stirred an additional 48 hr.Removal of the majority of the solvent in vacuo afforded a yellow solidwhich was filtered and dried to give the tetracyclic ketal (3.62 g, 92%,mp 215°-218° C.) which was used directly in the next step.Recrystallization of a portion of this material from CH₃ OH/CHCl₃ gavethe analytically pure material : mp 227°-229° C.

A mixture of THF (500 mL) and 30% HCl (180 mL) was cooled to 0° C., and3.62 g of the above ketal was added. This mixture was then stirred for24 hr at room temperature, after which TLC analysis snowed no cnange.Additional THF (1200 mL) and concentrated HCl (15 mL) were added, andthe solution was warmed to 40° C., whereupon it became homogeneous.After stirring for 48 hr at room temperature, TLC analysis indicatedcomplete hydrolysis of the ketal. The reaction mixture was thenconcentrated in portions at room temperature, and the resulting slurrywas extracted with CHCl₃ (3×100 mL), washed with brine, and dried.Concentration and drying in vacuo gave4-fluoro-4-demethoxy-11-methoxy-11-deoxydaunomycinone (3.0 g, 91%). Thismaterial was used directly in the next step.

To a -78° C. solution of the above material (3.0 g) in CH₂ Cl₂ (630 mL)was added BCl₃ (80 mL of a 1 M solution in CH₂ Cl₂). The resulting darkpurple solution was stirred for 2 hr at -78° C. The reaction wasquenched with CH₃ OH, the solvent was removed in vacuo, and theresulting solid was dried overnight in vacuo. Tnis material wasdissolved in a boiling mixture of CHCl₃ /CH₃ OH (ca. 1:1), and thesolution was heated to reflux for 1 hr. Cooling and concentration invacuo produced a voluminous red/orange solid which was filtered anddried in vacuo to give in three crops 4-fluoro-4-demethoxydaunomycinone(2.39 g, 83%), mp 238°-241° C.

SYNTHESIS EXAMPLE 16 (±)1-Fluoro-4-demethoxydaunomycinone

To a 0° C. solution of DMSO (13 mL) and THF (13 mL) was added CH₃ Li(3.3 mL of a 1.29 M solution). After 5 min,4-fluoro-3-cyano-1(3H)-isobenzofuranone (0.75 g, 0.42 mmol) in DMSO (13mL) was added, giving a yellow/brown solution. After an additional 2min, the monoketal (1.43 g, 3.26 mmol) in THF (11.3 mL) was addedrapidly, whereupon tne solution turned a deep red color. The reactionmixture was allowed to stir at room temperature for 2 hr, and then thereaction was quenched by the addition of 5% HCl (40 mL). Tne majority ofthe THF was removed in vacuo, (CH₃)₂ CO (100 mL) was added, and thesolution was allowed to stir for 12 hr. The heterogenous solution wasthen cooled in an ice bath and filtered. The resulting orange/yellowsolid (1.19 g, 81% crude) was used directly in the next step.

A mixture of THF (125 mL) and concentrated HCl (18 mL) was cooled to 0°C., and the above ketal (1.10 g) added. This mixture was then stirredfor 24 nr at room temperature, and the resulting homogeneous mixture wasdiluted with water (35 mL). The acid was neutralized by cautiousaddition of solid Na₂ CO₃. The aqueous layer was then separated andextracted with CHCl₃ (2×30 mL), and the combined organic phase wasconcentrated in vacuo. This material was dissolved in CHCl₃, dried, andconcentrated to give a dark residue which crystallized after addition ofCH₃ OH to give an orange powder (0.77 g, 77%, mp 157°-159° C.). Thismaterial was used directly in the next step.

To a -78° C. solution of the above material (0.77 g) in CH₂ Cl₂ (160 mL)was added BCl₃ (19.3 mL) of a 1 M solution in CH₂ Cl₂. The resultingdark purple solution was stirred for 2 hr at -78° C. The reaction wasquenched with CH₃ OH (90 mL), the solvent removed in vacuo, and tneresulting solid dried overnight in vacuo. Recrystallization of thismaterial from CHCl₃ /CH₃ OH gave (±)1-fluoro-4-demethoxydaunomycinone(0.48 g, 66%), mp 205°-208° C.

SYNTHESIS EXAMPLE 17 (±)1,4-Difluoro-4-demethoxydaunomycinone

To a 0° C. solution of DMSO (22.5 mL) and THF (22.5 mL) was added CH₃ Li(6.2 mL of a 1.29 M solution). After 5 min,4-fluoro-3-cyano-1(3H)-isobenzofuranone (1.5 g., 7.7 mmol) in DMSO (22.5mL) was added, giving a golden brown solution. After an additional 2min, the monoketal (2.6 g, 5.9 mmol) in THF (22.5 mL) was added rapidly,whereupon the solution turned a deep red color which took on ared/violet hue after 15 min. The reaction mixture was allowed to stir atroom temperature for 2 hr, and then the reaction was quenched by theaddition of 5% HCl (50 mL). The majority of the THF was removed invacuo, (CH₃)₂ CO (100 mL) was added, and the solution was allowed tostir for 12 hr. The solvents were removed in vacuo, and the resultingbrown oil was dissolved in CH₂ C₁₂ (500 mL) and washed with an equalvolume of water (3×). Concentration and drying afforded a dark oil whichwas used directly in the next step.

A mixture of THF (500 mL) and 30% HCl (200 mL) was cooled to 0° C., andthe above ketal was added. This mixture was then stirred for 3 days atroom temperature. The reaction mixture was concentrated in vacuo toabout one third of its original volume and then diluted with an equalvolume of water. This mixture was then extracted with CHCl₃ (2×250 mL),and the CHCl₃ layer was washed with an equal volume of water. Standardworkup and drying under vacuo for 12 hr gave a reddish brown foam whichwas used directly in the next step.

To a -78° C. solution of the above material (2.1 g) in CH₂ Cl₂ (250 mL)was added BCl₃ (80 mL of a 1 M solution in CH₂ Cl₂) over a period of 0.5hr. The resulting royal purple solution was stirred for 2 hr at -70° C.,and then the reaction was quenched with CH₃ OH (375 mL). Concentrationgave a dark oil which was dissolved in CHCl₃ and washed with water toremove residual HCl. After concentration, the mixture was redissolved inCHCl₃ /CH₃ OH (200:100 mL), and the solution was heated to reflux for 1hr. Concentration to ca. 50 mL gave a beautiful red solid (0.78 g),showing an analytically pure sample by 500-MHz ¹ H-NMR. This materialwas homogeneous by TLC but showed a wide melting-point range, 110°-125°C. The mother liquors were chromatographed on silica gel (1% CH₂ Cl₂/CH₃ OH as eluant) to afford an additional 0.78 g of slightly less purematerial as judged from the 500-MHz ¹ H NMR. The total yield of materialacceptable for coupling was 1.3 g (52% overall from monoketal).

SYNTHESIS EXAMPLE 184'-O-Acetyl-3'-N-trifluoroacetyl-4-demethoxy-4-fluorodaunomycin

To a solution of (+)4-Demethoxy-4-fluorodaunomycinone (50.3 mg, 0.125mmol) in dichloromethane (7 mL) was added mercuric bromide (50 mg),mercuric oxide (209 mg) and molecular sieves 4Å (0.7 g). To thisprepared suspension a solution of4-O-acetyl-2,3,6-trideoxy-3-N-trifluoroacetyl-α-L-lyxo-hexopyranopylchloride (76.6 mg, 0.28 mmol) in dichloromethane (6 mL) was added.Reaction was stopped when thin layer cnromatography(toluene:acetone/6:1) showed no aglycone in the reaction mixture.Filtration gave a red solution that was washed with 30% KI and water,then dried with MgSO₄ and after filtration evaporated. Crystallizationfrom acetone:hexane gave a red solid; yield 70.6 mg: mp. 150°-152° C.,[α]_(D) ²² +132° C. (c 0.05, CHCl₃).

SYNTHESIS EXAMPLE 19 4-Demetnoxy-4-fluorodaunomycin Hydrochloride

4'-O-Acetyl-3'-N-trifluoroacetyl-4-demetnoxy-4-fluorodaunomycin (50 mg,0.076 mmol) was dissolved in methanol and treated with sodium methoxidein methanol (0.57 mL of 0.135 M solution). The reaction was stopped byadding Dry Ice, poured into an excess of dichloromethane, and washedthree times with water. The organic layer was dried with MgSO₄ and thenfiltered. Evaporation gave a red solid. The resultant 4'-hydroxyderivative was treated with 0.1 M sodium hydroxide (10 mL) for 25 min.Extraction with dichlormethane and dichlormethane-methanol mixture gavea red solution that was extracted with 5% nydrochloric acid solution.The solution was adjusted with base to pH 9 and washed again withdichloromethane and a dicnloromethanemethanol mixture. The organic layerwas dried with MgSO₄, filtered and evaporated to dryness. Thin layerchromatography (chloroform:methanol/2:1) showed the presence of oneproduct. The red solid was redissolved in methanol (1 mL) and treatedwith methanolic hydrochloride solution in methanol (0.5 mL, 0.28 M).Addition of an excess of ethyl ether caused the precipitation of a redsolid (9.2 mg) which was filtered off and dried.

The same coupling method was used to prepare4'-O-acetyl-3'-N-trifluoroacetyl-4-demethoxy-1,4-difluorodaunomycin withthe natural 7S,9S configuration (mp 153°-154° C., [α]_(D) ²³ +175° (C0.05, CHCl₃) and its 7R,9R counterpart (mp 154°-155° C., [α]_(D) ²³-365° (C 0.005, CHCl₃)).

SYNTHESIS EXAMPLE 20(7S,9S)-4-Demethoxy-4-fluoro-7-0-(3,4-di-O-acetyl-2,6-dideoxy-2-iodo-.alpha.-L-manno-hexopyranosyl)daunomycinone and (7R,9R)-4-demethoxy-4-fluoro-7-0-(3,4-di-O-acetyl-2,6-dideoxy-2-iodo-α-L-manno-hexopyranosyl)daunomycinone

±4-demethoxy-4-Fluorodaunomycinone (311 mg, 0.806 mmol) was dissolved in6 mL of acetonitrile and then 3,4-di-O-acetyl-L-rhamnal (259 mg) inoxolane (3 mL) was added. To the vigorously-stirred reaction mixture,N-iodosuccinimide (326 mg) was added and the mixture was left at roomtemperature overnight. After 25 hr, additional portions of3,4-di-O-acetyl-L-rhamnal (130 mg) and N-iodosuccinimide (170 mg) wereadded. After 4 hr, the reaction mixture was diluted with dicnloromethaneand washed with 10% aqueous sodium thiosulfate and water. Drying witnMgSO₄, filtration and evaporation gave a red solid. Combination ofappropriate fractions from tnree successive chromatographic separations(toluene:acetone/9:1) gave after crystallization(dichloromethane:hexane) tne (7S,9S) (108.9 mg) and (7R,9R) (120.7 mg)compounds. 7S,9S:mp 133°-135°, [α]_(D) ²⁷ -26° (C 0.02, CHCl₃).

Using the same procedure, tne compounds(7S,9S)-4-demetnoxy-1-fluoro-7-O-(3,4-di-O-acetyl-2,6-dideoxy-2-iodo-.alpha.-L-mannopyranosyl)daunomycinone[mp 128°-129°, ¹ H NMR 5.73 (H-1'), 5.25 (H-7), 5.19 (H-4')];(7S,9S)-4-demethoxy1,4-difluoro-7-O-(3,4-di-O-acetyl-2,6-dideoxy-2-iodo-.alpha.-L-mannopyranosyl)daunomycinone[mp 142°-144°, [α]_(D) ²³ +23° (C 0.06 CHCl₃)] and its 7R, 9R analogue[mp 138°-141°, [α]_(D) ²³ -270° (C 0.05, CHCl₃)] were prepared.

SYNTHESIS EXAMPLE 21(7S,9S)-4-Demethoxy-1-fluoro-7-O-(2,6-oideoxy-2-iodo-α-L-manno-hexopyranosyl)daunomycinone

(7S,9S)-4-demethoxy-1-fluoro-7-O-(3,4-di-O-acetyl-2,6-dideoxy-2-iodo-.alpha.-L-mannopyranosyl)daunomycinone(55.8 mg, 0.077 mmol) was dissolved in methanol (200 mL) and then sodiummethoxide (2.5 mL of 0.371 M solution) was added. The reaction wasstopped by adding Dry Ice, diluting with dichlorometnane and washingwith water. The organic layer was dried with Na₂ SO₄, filtered andevaporated. The obtained solid was crystallized fromacetone:dichloromethane:hexane to give 28.2 mg of red precipitate (mp132°-135°).

BIOLOGICAL EXAMPLE

Groups of mice were inoculated by intraperitoneal injection with thelymphocytic leukemia cell line P-388. On day 1, 24 hours afterimplantation of the tumor cells, groups of test mice were administered asingle intraperitoneal does of Compounds 1 to 4 below. For comparison,similar groups of mice were challenged with the P-388 tumor cells andgiven a single dose of daunomycin on day 1 following implantation of thetumor cells. The animals were observed and their survival compared withthat of control animals which received the same tumor inoculation butwere not treated with the drug. The survival ratio T/C, a ratio of thetest Median Survival Time (MST) relative to control MST was determined.An increase in the T/C indicates an increase in the antitumor activityof the compound. If T/C is less than 100, the dosage is consideredtoxic. A T/C of 125 is indicative of antitumor activity.

Compound1,3'-deamino-4-demethoxy-3',4'-di-O-acetyl-4-fluoro-2'-iododaunomycin[4-Demethoxy-4-fluoro-7-O-(3,4-di-O-acetyl-2,6-dideoxy-2-iodo-α-L-mannopyranosyl)daunomycinone]

Compound 2 3'-deamino-4-demethoxy-4-fluoro-2'-iodo-daunomycin[4-Demethoxy-4-fluoro-7-O-(2,6-dideoxy-2-iodo-α-L-mannopyranosyl)daunomycinone]

Compound 3 4-demethoxy-4-fluorodaunomycin

Compound 4 3'-deamino-4-demethoxy-1-fluoro-2'-iododaunomycin[4-Demethoxy-1-fluoro-7-O-(2,6-dideoxy-2-iodo-α-L-mannopyranosyl)daunomycinone]

Control daunomycin

The survival ratios are shown below:

    ______________________________________                                        Dosage                 % Increase                                             (mg/kg)         T/C    In Life                                                ______________________________________                                        Compound 1                                                                    60               79    --                                                     30               89    --                                                     15              237    137                                                    7.5             177    77                                                     3.75            145    45                                                     Compound 2                                                                    30               49    --                                                     20               59    --                                                     10               74    --                                                     5               257    157                                                    2.5             211    111                                                    Compound 3                                                                    4                55    --                                                     2                70    --                                                     1               102    --                                                     0.5             165    65                                                     Compound 4                                                                    34               75    --                                                     17              220    120                                                    8.5             173    73                                                     4.25            135    35                                                     2.13            131    31                                                     1.06            121    --                                                     0.531           115    --                                                     0.266            98    --                                                     Control                                                                       20               54    --                                                     10              110    --                                                     5               163    63                                                     ______________________________________                                    

The results given in the table show that at non-toxic doses the ratio ofsurviving treated animals to surviving untreated animals increasessignificantly.

Having described the invention in detail and by reference to specificembodiments thereof, it will be apparent that numerous modifications andvariations are possible without departing from the scope of thefollowing claims.

What is claimed is:
 1. A compound of the formula (I) ##STR11## where Ris hydrogen or hydroxyl, one of X and Y is fluorine and the other ishydrogen or both X and Y are fluorine, and S_(u) is a hydrogen atom or asugar moiety represented by the formula ##STR12## wherein one of R¹ andR² is hydrogen and the other is hydroxy, acyloxy having 2 to 4 carbonatoms or a halogen atom; one of R³ and R⁴ is hydrogen and the other isamino; and R⁵ and R⁶ are hydrogen; and pharmaceutically acceptable acidaddition salts thereof.
 2. A compound according to claim 1 wherein S isdaunosamyl.
 3. A compound according to claim 2 wherein X is hydrogen andY is fluorine.
 4. A compound according to claim 2 wherein X is fluorineand Y is hydrogen.
 5. A compound according to claim 2 wherein X and Yare fluorine.
 6. A compound according to claim 2 wherein Su is acosamyl.7. A compound according to claim 6 wherein X is hydrogen and Y isfluorine and R is hydroxyl.
 8. A compound according to claim 1 whereinone of R¹ and R² is a halogen atom.
 9. The compound according to claim 8wherein R¹ is a halogen atom.
 10. The compound according to claim 9wherein R¹ is an iodine atom.
 11. The compound of claim 10 wherein X ishydrogen and Y is fluorine.
 12. The compound of claim 11 wherein R ishydroxyl.
 13. The compound of claim 1 wherein Su is a hydrogen atom.